Ubiquitin is a small protein consisting of 76 amino acids that is important in the regulation of protein function in the cell. Ubiquitination and deubiquitination are enzymatically mediated processes by which ubiquitin is covalently bound to or unbound from a target protein. These processes have been implicated in the regulation of the cell cycle, apoptosis, the marking of transmembrane proteins such as receptors for removal, regulation of DNA transcription and repair, and other important functions. Proteins are targeted for degradation by the proteasome in the cell by being “tagged” with three or more ubiquitin molecules (polyubiquitination). The binding of a single ubiquitin molecule (monoubiquitination) does not generally target the monoubiquitinated protein for degradation. Rather, it may trigger activities such as DNA repair and gene silencing, among other functions. Huang and D'Andrea (2006) Mol Cell Biol. 7:323-34.
Deubiquitination allows ubiquitin to be recycled and restores the function of the deubiquitinated proteins. Ubiquitin molecules are cleaved from a protein by deubiquitinating enzymes, which are cysteine proteases that operate through an active site thiol. There are approximately 95 different deubiquitinating enzymes in human cells. Huang et al. (2006) Nature Cell Biol. 8(4):339-47. Among them, Ubiquitin Specific Protease 1 (USP1), also known as ubiquitin specific peptidase 1 and as ubiquitin carboxyl terminal hydrolase 1, has been found to regulate the repair of DNA damage induced by DNA cross-linking agents, which include agents such as cisplatin, mitomycin C (MMC), diepoxybutane (DEB), ionizing radiation (IR), and ultraviolet (UV) radiation.
USP1 has been shown to deubiquitinate monoubiquitinated Fanconi anemia group complementation group D2 (FANCD2-Ub), a protein that in its monoubiquitinated form mediates DNA repair from the damage induced by the aforementioned agents. Nijman et al. (2005) Mol Cell 17:331-39. USP1 also has been shown to deubiquitinate monoubiquitinated proliferating cell nuclear antigen (PCNA-Ub), a protein that in its monoubiquitinated form activates DNA translesion synthesis, a polymerase-mediated bypass of DNA lesions. Huang et al. (2006) Nature Cell Biol. 8(4):339-47.
Recently it was reported that USP1 forms a complex with and is activated by USP1 associated factor 1 (UAF1), also known as WD repeat domain 48. Cohn et al. (2007) Mol Cell 28:786-97; US 2008/0167229 A1. The active USP1/UAF1 complex controls cellular levels of monubiquitinated FANCD2. Even more recently, it was reported that UAF1 also separately forms complexes with and activates two additional deubiquitinating enzymes, ubiquitin specific protease 12 (USP12) and ubiquitin specific protease 46 (USP46). Cohn et al. (2009) J Biol Chem. 284(8):5343-51.
Co-owned US 2008/0167229 discloses three compounds, β-lapachone, Biomol AP401 (propidium iodide), and RK-682, as potential small molecule inhibitors of USP1/UAF1 complex-mediated deubiquitinase activity.